Capsaicinoids are products of the phenylpropanoid pathway, a central and ubiquitous pathway in plant secondary metabolism. The products of this pathway are diverse, perform a multitude of functions in the plant, and are the chemical basis for numerous pharmaceutical agents. There are heritable differences in the amount and type of capsaicinoids that accumulate in fruit of different Capsicum lines; these differences are controlled by other genes, whose nature is also unknown. Our long-term goal is to understand the genetic, biochemical, physiological and environmental influences on the biosynthesis of capsaicinoids. We have demonstrated that there is differential transcription of the biosynthetic genes for capsaicinoids in the placenta of Capsicum spp. The pattern of transcript accumulation suggests that there are transcription factors that will bind to common cis elements in the promoters of these genes. None of the structural genes that we have characterized appear to be associated with the loci that control pungency, either the C locus or the QTL locus, caps. We anticipated that the genes that control pungency are likely regulatory, and now we are in a position to formally test that hypothesis. This SCORE competing renewal proposal outlines a series of experiments to isolate and characterize candidate transcription factors. We propose to characterize the cis-elements of the structural genes as well as candidate regulatory genes for the capsaicinoid pathway. The specific aims of this proposal are: 1) Complete a detailed characterization of the promoter regions of the structural genes on the capsaicinoid biosynthetic pathway; 2) Isolate and characterize genes for transcription factors expressed uniquely in chile placenta; 3) Determine the cell specific pattern of expression for these transcription factors using immunolocalization; 4) Determine if these transcription factors regulate/bind to promoters of capsaicionoid biosynthetic genes. To meet these objectives we will isolate placental specific transcription factors from habanero using a combination of cDNA library screening and PCR amplification. We have a bZIP candidate in hand and expect to isolate representative members of the myb and bHLH classes. Those trans-factors that are localized for accumulation in the nucleus of placental epidermal cells will be used in in vitro (gel shift) and in vivo (chromatin immunoprecipitation) assays to demonstrate binding.